scholarly journals Induce Pluripotency via Specific distal enhancer-promoter associations

2021 ◽  
Author(s):  
Xiusheng Zhu ◽  
Lei Huang ◽  
Dongwei Li ◽  
Jing Luo ◽  
Qitong Huang ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
De Novo ◽  
Induced Pluripotent Stem Cell ◽  
Marker Genes ◽  
Genomic Information ◽  
Distal Enhancer ◽  
Self Renewal ◽  
Induced Pluripotent ◽  
Pluripotency Maintenance

Induced pluripotent stem cell(iPSC) technology promises to be an inexhaustible source of any type of cell needed for therapeutic and research purposes.It is unclear that how distal enhancer-promoter associations/3D chromatin conformation involving in the capacity of self-renewal and pluripotency maintenance. In this study, we have selected a few defined enhancer-promoter associations. After screening of enhancer specificity and activity individually, we design the different combinations and transfect these enhancers into the MEF cells. We simultaneously transfect 7 determined enhancers which represents various specific distal chromatin associations into a GFP tracing MEF cell line. We observe that the MEF cells start generating iPS-like clones at day 22. Importantly, our validations with three germ layer marker genes and in vitro experiments have further confirmed the pluripotency of these clones. Here, our study proposes a potential de novo method of a low-genetic risk iPS generation by introducing spatiotemporal distal chromatin associations. This result also paves out the way on utilizing 3D genomic information to alter cell identity and reprogramming for potential therapeutic strategy.

scholarly journals Shaking culture enhances chondrogenic differentiation of mouse induced pluripotent stem cell constructs

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Phoonsuk Limraksasin ◽  
Yukihiro Kosaka ◽  
Maolin Zhang ◽  
Naohiro Horie ◽  
Takeru Kondo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Wnt Signaling ◽  
Pluripotent Stem Cell ◽  
Chondrogenic Differentiation ◽  
Induced Pluripotent Stem Cell ◽  
Immunohistochemical Analysis ◽  
Cell Aggregation ◽  
Marker Genes ◽  
Induced Pluripotent

Abstract Mechanical loading on articular cartilage induces various mechanical stresses and strains. In vitro hydrodynamic forces such as compression, shear and tension impact various cellular properties including chondrogenic differentiation, leading us to hypothesize that shaking culture might affect the chondrogenic induction of induced pluripotent stem cell (iPSC) constructs. Three-dimensional mouse iPSC constructs were fabricated in a day using U-bottom 96-well plates, and were subjected to preliminary chondrogenic induction for 3 days in static condition, followed by chondrogenic induction culture using a see-saw shaker for 17 days. After 21 days, chondrogenically induced iPSC (CI-iPSC) constructs contained chondrocyte-like cells with abundant ECM components. Shaking culture significantly promoted cell aggregation, and induced significantly higher expression of chondrogenic-related marker genes than static culture at day 21. Immunohistochemical analysis also revealed higher chondrogenic protein expression. Furthemore, in the shaking groups, CI-iPSCs showed upregulation of TGF-β and Wnt signaling-related genes, which are known to play an important role in regulating cartilage development. These results suggest that shaking culture activates TGF-β expression and Wnt signaling to promote chondrogenic differentiation in mouse iPSCs in vitro. Shaking culture, a simple and convenient approach, could provide a promising strategy for iPSC-based cartilage bioengineering for study of disease mechanisms and new therapies.

Use of 3D culture systems to generate human induced pluripotent stem cell-derived [beta]-cells in vitro

2018 ◽  
Author(s):  
Fantuzzi Federica ◽  
Toivonen Sanna ◽  
Schiavo Andrea Alex ◽  
Pachera Nathalie ◽  
Rajaei Bahareh ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Cells ◽  
Pluripotent Stem Cell ◽  
3D Culture ◽  
Induced Pluripotent Stem Cell ◽  
Culture Systems ◽  
Induced Pluripotent

scholarly journals Bestrophinopathies: perspectives on clinical disease, Bestrophin-1 function and developing therapies

2021 ◽  
Vol 13 ◽  
pp. 251584142199719
Author(s):  
Simranjeet Singh Grewal ◽  
Joseph J. Smith ◽  
Amanda-Jayne F. Carr
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Incomplete Penetrance ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
High Acuity ◽  
Induced Pluripotent

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that typically affect the macular region, an area synonymous with central high acuity vision. This spectrum of disorders is caused by mutations in bestrophin1 ( BEST1), a protein thought to act as a Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE) of the eye. Although bestrophinopathies are rare, over 250 individual pathological mutations have been identified in the BEST1 gene, with many reported to have various clinical expressivity and incomplete penetrance. With no current clinical treatments available for patients with bestrophinopathies, understanding the role of BEST1 in cells and the pathological pathways underlying disease has become a priority. Induced pluripotent stem cell (iPSC) technology is helping to uncover disease mechanisms and develop treatments for RPE diseases, like bestrophinopathies. Here, we provide a comprehensive review of the pathophysiology of bestrophinopathies and highlight how patient-derived iPSC-RPE are being used to test new genomic therapies in vitro.

In vitro pharmacologic testing using human induced pluripotent stem cell-derived cardiomyocytes

2009 ◽  
Vol 385 (4) ◽  
pp. 497-502 ◽  
Author(s):  
Tomofumi Tanaka ◽  
Shugo Tohyama ◽  
Mitsushige Murata ◽  
Fumimasa Nomura ◽  
Tomoyuki Kaneko ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

scholarly journals Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabriel Peinkofer ◽  
Martina Maass ◽  
Kurt Pfannkuche ◽  
Agapios Sachinidis ◽  
Stephan Baldus ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Developmental Stage ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

Abstract Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

scholarly journals Disease Modeling and Disease Gene Discovery in Cardiomyopathies: A Molecular Study of Induced Pluripotent Stem Cell Generated Cardiomyocytes

2021 ◽  
Vol 22 (7) ◽  
pp. 3311
Author(s):  
Satish Kumar ◽  
Joanne E. Curran ◽  
Kashish Kumar ◽  
Erica DeLeon ◽  
Ana C. Leandro ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Disease Gene ◽  
Disease Modeling ◽  
Gene Discovery ◽  
Induced Pluripotent Stem Cell ◽  
P Value ◽  
Disease Gene Discovery ◽  
Induced Pluripotent

The in vitro modeling of cardiac development and cardiomyopathies in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) provides opportunities to aid the discovery of genetic, molecular, and developmental changes that are causal to, or influence, cardiomyopathies and related diseases. To better understand the functional and disease modeling potential of iPSC-differentiated CMs and to provide a proof of principle for large, epidemiological-scale disease gene discovery approaches into cardiomyopathies, well-characterized CMs, generated from validated iPSCs of 12 individuals who belong to four sibships, and one of whom reported a major adverse cardiac event (MACE), were analyzed by genome-wide mRNA sequencing. The generated CMs expressed CM-specific genes and were highly concordant in their total expressed transcriptome across the 12 samples (correlation coefficient at 95% CI =0.92 ± 0.02). The functional annotation and enrichment analysis of the 2116 genes that were significantly upregulated in CMs suggest that generated CMs have a transcriptomic and functional profile of immature atrial-like CMs; however, the CMs-upregulated transcriptome also showed high overlap and significant enrichment in primary cardiomyocyte (p-value = 4.36 × 10−9), primary heart tissue (p-value = 1.37 × 10−41) and cardiomyopathy (p-value = 1.13 × 10−21) associated gene sets. Modeling the effect of MACE in the generated CMs-upregulated transcriptome identified gene expression phenotypes consistent with the predisposition of the MACE-affected sibship to arrhythmia, prothrombotic, and atherosclerosis risk.

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